Tuberculosis is the leading cause of death in the world with an estimated 9 million new cases of tuberculosis and 2.9 million deaths occurring from the disease each year. In the United States, the steadily declining incidents of tuberculosis has been reversed since 1985. This problem is compounded by the increasing incidence of drug-resistant strains of Mycobacterium tuberculosis.
Recent outbreaks of tuberculosis have involved settings in which a large number of HIV-infected persons resided in close proximity (e.g., AIDS wards in hospitals, correctional facilities, and hospices). Transmission of tuberculosis to health care workers occurred in these outbreaks; 18 to 50% of such workers showed a conversion in their skin tests. See F. Laraque et. al., "Tuberculosis in HIV-Infected Patients," The AIDS Reader (September/October 1992), which is hereby incorporated by reference.
There are two basic clinical patterns that follow infection with Mycobacterium tuberculosis.
In the majority of cases, inhaled tubercle bacilli ingested by phagocytic alveolar macrophages are either directly killed or grow intracellularly to a limited extent in local lesions called tubercles. Infrequently in children and immunocompromised individuals, there is early hematogenous dissemination with the formation of small miliary (millet-like) lesions or life-threatening meningitis. More commonly, within 2 to 6 weeks after infection, cell-mediated immunity develops, and infiltration into the lesion of immune lymphocytes and activated macrophages results in the killing of most bacilli and the walling-off of this primary infection, often without symptoms being noted by the infected individual. Skin-test reactivity to a purified protein derivative ("PPD") of tuberculin and, in some cases, X-ray evidence of a healed, calcified lesion provide the only evidence of the infection. Nevertheless, to an unknown extend, dormant but viable Mycobacterium tuberculosis bacilli persist.
The second pattern is the progression or breakdown of infection to active disease. Individuals infected with Mycobacterium tuberculosis have a 10% lifetime risk of developing the disease. In either case, the bacilli spread from the site of initial infection in the lung through the lymphatics or blood to other parts of the body, the apex of the lung and the regional lymph node being favored sites. Extrapulmonary tuberculosis of the pleura, lymphatics, bone, genito-urinary system, meninges, peritoneum, or skin occurs in about 15% of tuberculosis patients. Although many bacilli are killed, a large proportion of infiltrating phagocytes and lung parenchymal cells die as well, producing characteristic solid caseous (cheese-like) necrosis in which bacilli may survive but not flourish. If a protective immune response dominates, the lesion may be arrested, albeit with some residual damage to the lung or other tissue. If the necrotic reaction expands, breaking into a bronchus, a cavity is produced in the lung, allowing large numbers of bacilli to spread with coughing to the outside. In the worst case, the solid necrosis, perhaps a result of released hydrolases from inflammatory cells, may liquefy, which creates a rich medium for the proliferation of bacilli, perhaps reaching 10.sup.9 per milliliter. The pathologic and inflammatory processes produce the characteristic weakness, fever, chest pain, cough, and, when a blood vessel is eroded, bloody sputum.
Two of the major antimicrobial mechanisms of activated macrophages depend on the synthesis of inorganic radical gases by immunologically regulated flavocytochrome complexes that use NADPH to reduce molecular oxygen. When oxygen is the sole co-substrate, the product is superoxide (O.sub.2 --)(Nathan, et al., "Mechanisms of Macrophage Antimicrobial Activity," Trans. R. Soc. Trop. Med. Hyg., 77:620-30 (1983)); when L-arginine is an additional co-substrate, the product is nitric oxide (NO)(Nathan, et al., "Role of Nitric Oxide Synthesis in Macrophage Antimicrobial Activity," Curr. Opin. Immunol., 3:65-70 (1991)). These radicals react with oxygen, transition metals, halides, sulfhydryls, and each other to produce a series of broadly cytotoxic products termed reactive oxygen intermediates ("ROI") and reactive nitrogen intermediates ("RNI"), as well as at least one compound with features of both, peroxynitrite (OONO--)(Butler, et al., "NO, Nitrosonium Ions, Nitroxide Ions, Nitrosothiols and Iron-Nitrosyls in Biology: A Chemist's Perspective," Trends Pharmacol. Sci., 16:18-22 (1995) and DeGroote, et al., "NO Inhibitions: Antimicrobial Properties of Nitric Oxide," Clin. Infect. Dis., 21:S162-5 (1995)).
Mycobacterium tuberculosis resist ROI by a diversity of mechanisms. Phenolic glycolipids (Neill, et al., "The Effect of Phenolic Glycolipid-1 From Mycobacterium Leprae on the Antimicrobial Activity of Human Macrophages," J. Exp. Med., 167:30-42 (1988)) and cylcopropanated mycolic acids (Sherman, et al., "Disparate Responses to Oxidative Stress in Saprophytic and Pathogenic Mycobacteria," Proc. Natl. Acad. Sci. USA, 92:6625-9 (1995)) protect the cell wall, while catalase, alkylhydroperoxide reductase (Sherman, et al., "Compensatory ahpC Gene Expression in Isoniazid-Resistant Mycobacterium Tuberculosis," Science, 272:1641-3 (1996)) and superoxide dismutase (Dumarey, et al., "Selective Mycobacterium Avium-Induced Production of Nitric Oxide by Human Monocyte-Derived Macrophages," J. Leuk. Biol., 56:36-40 (1994) and Zhang, et al., "Alterations in the Superoxide Dismutase Gene of an Isoniazid-Resistant Strain of Mycobacterium Tuberculosis," Infect. Immun., 60:2160-5 (1992)) guard the cytosol. Moreover, Mycobacterium tuberculosis may enter macrophages via complement receptors (Chan, et al., "Immune Mechanisms of Protection. In Tuberculosis: Pathogenesis, Protection and Control," B. R. Bloom, ed. (Washington:ASM), 389-415 (1994) and Schlesinger, et al., "Phagocytosis of Mycobacterium Tuberculosis is Mediated by Human Monocyte Complement Receptors and Complement Component C3," Immunol., 144:2271-80 (1990)), a pathway that fails to stimulate generation of ROI in some populations of macrophages (Wright, et al., "Receptors for C3b and C3bi Promote Phagocytosis But Not the Release of Toxic Oxygen From Human Phagocytes," J. Exp. Med., 158:2016-2023 (1983)). The ability of Mycobacterium tuberculosis to mount such a broad defense against ROI implies that other products of the activated macrophage may be more important for tuberculostasis. Indeed, activated murine macrophages selectively deficient in production of ROI were nonetheless mycobactericidal (Chan, et al., "Killing of Virulent Mycobacterium Tuberculosis by Reactive Nitrogen Intermediates Produced by Activated Murine Macrophages," J. Exp. Med., 175:1111-1122 (1992)). Not all mechanisms of defense against reactive oxygen intermediates are known.
In contrast, abundant evidence establishes the importance of RNI in the control of mycobacteria, at least in the mouse. Mycobacterium tuberculosis proliferates exuberantly in mice rendered selectively deficient in nitric oxide synthase type 2 (NOS2; iNOS). The organism also grows rapidly in mice made deficient in components of the cell-mediated immune response that normally leads to the induction of NOS2, as well as in mice dosed with organochemicals (Chan, et al., "Effects of Protein Malnutrition on Tuberculosis in Mice," Proc. Natl. Acad. Sci. USA, 93:14857-14861 (1996)) or glucocorticoids that inhibit the action or expression of NOS2. NOS2 is present in macrophages collected from the lungs of patients with tuberculosis (Nicholson, et al., "Inducible Nitric Oxide Synthase in Pulmonary Alveolar Macrophages From Patients With Tuberculosis," J. Exp. Med., 183:2293-302 (1996)), raising the possibility that the enzyme may play an antitubercular role in people as well as in mice.
Ignorance of the molecular basis of virulence and pathogenesis is great. It has been suggested that the establishment of molecular evidence regarding avirulent strains, the identification and cloning of putative virulence genes of the pathogen, and the demonstration that virulence can be conveyed to an avirulent strain by those genes is necessary. Although avirulent strains of Mycobacterium tuberculosis exist, the nature of the mutations is unknown.
There have been many prescribed treatment regimens for tuberculosis. The regimen recommended by the U.S. Public Health Service and the American Thoracic Society is a combination of isoniazid, rifampicin, and pyrazinamide for two months followed by administration of isoniazid and rifampicin for an additional four months. In persons with HIV infection, isoniazid and rifampicin treatment are continued for an additional seven months. This treatment, called the short-course chemotherapy, produces a cure rate of over 90% for patients who complete it. Treatment for multi-drug resistant tuberculosis requires addition of ethambutol and/or streptomycin in the initial regimen, or second line drugs, such as kanamycin, amikacin, capreomycin, ethionamide, cyclcoserin, PAS, and clofazimine. New drugs, such as ciprofloxacin and ofloxacin can also be used. For individuals infected with conventional Mycobacterium tuberculosis and showing PPD positive results, chemoprophylaxis with isoniazid has been about 90% effective in preventing the disease. Tuberculosis and these treatments are discussed in more detail in B. Bloom et al., "Tuberculosis: Commentary on a Reemergent Killer," Science, 257:1055-64 (1992); "Control of Tuberculosis in the United States," American Thoracic Society, 146:1623-33 (1992); City Health Information, vol. 11 (1992), which are hereby incorporated by reference.
There has been a recent resurgence of tuberculosis in the United States due to the emergence of Mycobacterium tuberculosis strains which are resistant to isoniazid. Contrary to previous hypothesis, the drug resistant character of most of these strains is not believed to be caused by a complete deletion in the katG gene which encodes for an enzyme having catalase-peroxidase activity. Stoeckle, et. al., "Catalase-Peroxidase Gene Sequences in Isoniazid-Sensitive and -Resistant Strains of Mycobacterium tuberculosis in New York City," J. Infect. Dis. 168: 1063.varies.65 (1993); Ferrazoli, et. al., "Catalase Expression, katG and MIC of Isoniazid for Mycobacterium tuberculosis Isolates from Sao Paulo, Brazil," J. Infect. Dis. 171: 237-40 (1995). It has since been hypothesized that another genetic locus, inhA, is the target for isoniazid action. Banerjee, et al., "inhA, a Gene Encoding a Target for Isoniazid and Ethionamide in Mycobacterium tuberculosis," Science 263: 227-30 (1994), but see Mdluli, et al., "Biochemical and Genetic Data Suggest that InhA is not the Primary Target for Activated Isoniazid in Mycobacterium tuberculosis," J. Infect. Dis. 174: 1085-90 (1996).
Although the currently used treatments for tuberculosis have a relatively high level of success, the need remains to improve the success rate for treating this disease. Moreover, in view of the ever-increasing level of Mycobacterium tuberculosis strains which are resistant to conventional treatment regimens, new types of treatment must be developed. In high tuberculosis endemic areas, both in the United States and abroad, such resistant strains are becoming increasing present.